A biological sample, such as a tissue biopsy is obtained for analysis. The analysis will be a molecular analysis to determine if a specific analyte is present within the sample. The presence or absence of an analyte is frequently used to determine a disease state, such as cancer. For successful analysis, the tissue is processed by fixation, embedding, and sectioning onto a microscope slide. In preparation for staining, the tissue is deparaffinzed, rehydrated, and treated by target retrieval to render the presumed analyte susceptible to staining. The samples are then stained by either ISH or IHC to render the presumed analyte colored and visible when examined under the microscope. Thus, the analyte is either present (colored) or absent (not colored), and the disease state of the tissue is then determined.
What is needed is a method for not only determining the presence or absence of a single analyte, but also the presence or absence of multiple analytes. It would be useful to have a method of chromogen layering, wherein a first chromogen and a first stain (color) are produced on a sample, specific for a first analyte, followed by a second chromogen and a second stain (color) being produced on the same sample, specific for a second analyte. In addition, if desired, by overlaying the second stain on top of the first stain, a unique third color is produced that is specific for a third analyte. Therefore, the distribution of different colors throughout the sample could be used to identify at least two or more analytes simultaneously within a single sample.